Neocentromere-based mini-chromosomes or artificial chromosomes

Abstract

The present invention is directed generally to a defined or isolated nucleic acid molecule encompassing a neocentreomere or a functional derivative thereof or a latent, synthetic or hybrid form thereof and its use inter alia in developing a range of eukaryotic mini-chromosomes and artificial chromosomes including mammalian (e.g. human) and non-mammalian mini-chromosomes and artificial chromosomes. The present invention further provides a telomere-associated chromosome truncation (TACT) approach to develop mini-chromosomes, but is not limited to this approach. It is directed to any truncation approach that produces a neocentromere-containing mini-chromosome, or any transfection approach using cloned or pre-fabricated DNA that provides neocentromere function in the construction of artificial chromosome. Such mini-chromosomes and artificial chromosomes are useful in a range of genetic therapies.

1 . An isolated nucleic acid molecule comprising a sequence of nucleotides derived from a eukaryotic chromosome and encompassing a neocentromere, which nucleic acid molecule exists in a truncated form in a compatible cell or, when introduced by transfection as a pre-fabricated DNA entity into a compatible cell, is capable of replicating, acting as an extra chromosomal element, and segregating with cell division.  
     
     
         2 . The isolated nucleic acid molecule of  claim 1  wherein the eukaryotic chromosome is derived from a mammal, including a human or primate, a plant, an avian species, an insect, a worm, a fungus, a yeast or a reptile.  
     
     
         3 . The isolated nucleic acid molecule of  claim 2  wherein the eukaryotic chromosome is derived from a human.  
     
     
         4 . The isolated nucleic acid molecule of  claim 2  wherein the eukaryotic chromosome is derived from a livestock animal.  
     
     
         5 . The isolated nucleic acid of  claim 1  comprising a q′ arm of the mardel(10) chromosome or its equivalent truncated by targeted telomere-associated truncation and further comprising the p′ arm of the mardel(10) chromosome or its equivalent truncated by random truncation.  
     
     
         6 . The isolated nucleic acid molecule of  claim 5  wherein said nucleic acid molecule comprises a neocentromere at a location equivalent to between q24 and q26 on chromosome 10 or its equivalent.  
     
     
         7 . The isolated nucleic acid molecule of  claim 6  wherein said nucleic acid molecule comprises a neocentromere at a location equivalent to at or about q25 on chromosome 10 or its equivalent.  
     
     
         8 . The isolated nucleic acid molecule of  claim 7  wherein said nucleic acid molecule comprises a neocentromere at a location equivalent to at or about q25.2 on chromosome 10 or its equivalent.  
     
     
         9 . The nucleic acid molecule of any one of claims  1 ,  6 ,  7  or  8  wherein the region corresponding to a neocentromere is substantially devoid of α-satellite DNA.  
     
     
         10 . The nucleic acid molecule of  claim 9  wherein the nucleic acid molecule is from about 0.5 to about 2.0 Mb in size.  
     
     
         11 . The nucleic acid molecule of  claim 10  wherein the nucleic acid molecule is from about 0.8 to about 1.6 Mb in size.  
     
     
         12 . A method for identifying a neocentromere or a functional homolog thereof, said method comprising isolating DNA by chromatin immunoprecipitation with an antibody specific to mammalian CENP-A and/or CENP-C or an antibody capable of cross interacting with mammalian CENP-A and/or CENP-C, amplifying the DNA isolated by immunoprecipitation, incorporating a label into the amplified DNA, probing a DNA array comprising genomic DNA or its equivalent with the labeled, amplified DNA, and identifying and isolating clones which hybridize to said immunoprecipitated DNA.  
     
     
         13 . The method of  claim 12  wherein the mammal is a human, livestock animal, companion animal or laboratory test animal.  
     
     
         14 . The method of  claim 13  wherein the mammal is a human.  
     
     
         15 . The method of  claim 14  wherein the neocentromere is at a location equivalent to between p24 and p26 on chromosome 10 or its equivalent.  
     
     
         16 . The method of  claim 15  wherein the neocentromere is at a location at or about q25 on chromosome 10 or its equivalent.  
     
     
         17 . The method of  claim 16  wherein the neocentromere is at a location at or about q25.2 on chromosome 10 or its equivalent.  
     
     
         18 . An isolated human neocentromere-based mini-chromosome (NC-MiC), said NC-MiC comprising a neocentromere or a latent, synthetic or hybrid form thereof which enables stable segregation during cell division.  
     
     
         19 . The isolated mini-chromosome of  claim 18  comprising a q′ arm of the mardel(10) chromosome or its equivalent truncated by targeted telomere-associated truncation and further comprising the p′ arm of the mardel(10) chromosome or its equivalent truncated by random truncation.  
     
     
         20 . The isolated mini-chromosome of  claim 19  wherein the neocentromere is at a location equivalent to between q24 and q26 on chromosome 10 or its equivalent.  
     
     
         21 . The isolated mini-chromosome of  claim 20  wherein the neocentromere is at a location at or about q25 on chromosome 10 or its equivalent.  
     
     
         22 . The isolated mini-chromosome of  claim 20  wherein the neocentromere is at a location at or about q25.2 on chromosome 10 or its equivalent.  
     
     
         23 . A method for generating a mini-chromosome, said method comprising: 
 introducing into a human or mammalian cell which carries a chromosome containing a neocentromere, a truncation construct comprising a vector having telomeric sequences, a selectable marker, homologous targeting DNA on one or other of the q′ or p′ arm of the target chromosome;    selecting for cells expressing the selectable marker;    introducing into said cells a second truncation construct comprising the other of said q′ or p′ targeting DNA;    selecting for cells expressing the selectable marker associated with said second truncation construct; and    then isolating the truncated chromosome which comprises a neocentromere.    
     
     
         24 . The method of  claim 23  wherein the cell is a human cell.  
     
     
         25 . The method of  claim 24  wherein the neocentromere is at a location equivalent to between q24 and q26 on chromosome 10 or its equivalent.  
     
     
         26 . The method of  claim 24  wherein the neocentromere is at a location at or about q25 on chromosome 10 or its equivalent.  
     
     
         27 . The method of  claim 24  wherein the neocentromere is at a location at or about q25.2 on chromosome 10 or its equivalent.  
     
     
         28 . An isolated cell comprising a chromosome having q′ and p′ arms flanking a neocentromere wherein one or both q′ and/or p′ arms are truncated.  
     
     
         29 . An isolated cell line deposited at ECAAC under Accession 00122001 (CHO/BE ZB30).  
     
     
         30 . An isolated cell line deposited at ECAAC under Accession 00122002 (HT1080-MIC 1).  
     
     
         31 . An isolated cell line deposited at ECAAC under Accession 00122003 (HT1080-MIC 2).  
     
     
         32 . An isolated cell line deposited at ECAAC under Accession 00122004 (HT1080-MIC 3).  
     
     
         33 . An isolated cell line deposited at ECAAC under Accession 00122005 (HT1080-MIC 4).  
     
     
         34 . An isolated cell line deposited at ECAAC under Accession 00122006 (HT1080-MIC 5).  
     
     
         35 . An isolated cell line deposited at ECAAC under Accession 00122007 (HT1080-MIC 5a).  
     
     
         36 . An isolated cell line deposited at ECAAC under Accession 00122008 (HT1080-MIC 5b).  
     
     
         37 . The mini-chromosome generated by the method of  claim 23 , wherein the mini-chromosome is an NC-MiC.  
     
     
         38 . The mini-chromosome generated by the method of  claim 23 , wherein the mini-chromosome is a YAC.  
     
     
         39 . The mini-chromosome generated by the method of  claim 23 , wherein the mini-chromosome is a HAC.  
     
     
         40 . The mini-chromosome generated by the method of  claim 23 , wherein the mini-chromosome is a MAC.  
     
     
         41 . The mini-chromosome generated by the method of  claim 23 , wherein the mini-chromosome is a PLAC.  
     
     
         42 . A method of gene transfer comprising introducing into a target cell the mini-chromosome of any one of claims  37 ,  38 ,  39 ,  40  or  41 .